大鼠耳蜗感觉上皮细胞的原代培养及其意义

The primary culture of rats cochlear sensory epithelial cell and the significance of expression of hair cell characteristic markers of CSEC

目的:建立大鼠耳蜗感觉上皮细胞(CSEC)的原代培养体系,为研究毛细胞再生的分子机制提供大量来源一致的细胞。方法:取出生1d(P1)的大鼠耳蜗感觉上皮(Corti器)作组织块培养,选择性消化抑制成纤维样细胞(FLC)生长;应用有限稀释法纯化CSEC,倒置显微镜下观察CSEC的形态、生长特征;应用细胞角蛋白18、波形蛋白、Brn3.a和Calretinin的免疫细胞化学及透射电镜等进行鉴定。BrdU标记CSEC核DNA观察CSEC有丝分裂活动。结果:培养第2天CSEC从组织块迁移出来,呈扁平、多角形、核大而圆的上皮细胞形态,细胞之间连接紧密,形成单层时呈“铺路石样”外观;FLC呈梭形“鱼群样”包绕CSEC生长,增殖速度快于CSEC。由纯化的CSEC构成的上皮单层可见由成百个CSEC包绕液体形成的dome。CSEC表达毛细胞的特征性标志物,证实培养的CSEC具有毛细胞前体细胞的特征;并通过有丝分裂活动产生新的细胞即毛细胞样细胞。结论:体外培养的CSEC表达毛细胞特征性标志物,具有毛细胞前体细胞的特征。CSEC原代培养体系的建立,为进一步探讨毛细胞再生的分子机制提供了实验条件。

Objective：To further investigate the mechanisms of hair cell generation or regeneration,the primary culture systems of cochlear sensory epithelial cell （CSEC） of rats were established. Method： Cochlear sensory epithelial （containing the organ of Corti） of postnatal day 1 （P1） rats was isolated by mechanical dissociation. The explants were grown on sterilized disposable plastic dishes, cultured in Dulbecco modified Eagle medium（DMEM）, and observed daily by inverted microscopy. The culture medium was changed twice a week. The pure CSEC was harvested by the limiting dilution method. CSEC was identified by immunocytochemical method with cytokeratin 18, vimentin, Brn3. a, Calretinin, BrdU and ultrastrctural examination with transmission electron microscopy. The markers of epithelial cell and the markers of hair cell were used to identify the origin and character of CSEC. CSEC mitotic division was detected by BrdU staining of nuclear DNA. Result：The fresh explants were light yellow. The morphology of CSEC couldn＇t be seen clearly under the inverted microscope because of the complex structures of cochlear sensory epithelial. CSEC grew out of the explants usually at 2nd culture day. Fibroblast like cells （FLC） around the CSEC grew faster than CSEC, but could easily be excluded. Pure CSEC may grow into monolayer with ＂cobblestone- like＂ appearance and show a large, flat, polygonal epithelial morphotype with big, round neuclei. Some cells showed ＂Dome＂ formation, probably due to fluid collection underneath the cell monolayer. The culture CSEC coexpressed cytokeratin 18 and vimentin has rich microvilli and complex tight junction,which indicated the epithelial origination of CSEC. Coexpressed of the Brn3. a and Calretinin of the hair cell characteristic markers suggested the culture cell may represent rat progenitor hair cell. BrdU staining showed CSEC produced new hair cell-like cell （progenitor hair cell） by the mitotic division. Conclusion：The primary culture systems of cochlear sensory epithelial cell of rats were successfully established in vitro. CSEC coexpressed the Characteristic markers of the immature hair cell,which identified the culture cell may represent progenitor cell of rats hair cell; It may be a suitable model for in-depth investigation the molecular mechanisms of hair cell generation or regeneration.